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The evaluation of the integrity and quantity of DNA extracted from archaeological human remains is a fundamental step before using the latest generation sequencing techniques in the study of evolutionary processes. Ancient DNA is highly fragmented and chemically modified; therefore, the present study aims to identify indices that can allow the identification of potentially amplifiable and sequenceable DNA samples, reducing failures and research costs. Ancient DNA was extracted from five human bone remains from the archaeological site of Amiternum L’Aquila, Italy dating back to the 9th–12th century and was compared with standard DNA fragmented by sonication. Given the different degradation kinetics of mitochondrial DNA compared to nuclear DNA, the mitochondrially encoded 12s RNA and 18s ribosomal RNA genes were taken into consideration; fragments of various sizes were amplified in qPCR and the size distribution was thoroughly investigated. DNA damage degree was evaluated by calculating damage frequency (λ) and the ratio between the amount of the different fragments and that of the smallest fragment (Q). The results demonstrate that both indices were found to be suitable for identifying, among the samples tested, those less damaged and suitable for post-extraction analysis; mitochondrial DNA is more damaged than nuclear, in fact, amplicons up to 152 bp and 253 bp, respectively are obtained.

Humans have always been exposed to tiny particles via dust storms, volcanic ash, and other natural processes, and our bodily systems are well adapted to protect us from these potentially harmful external agents. However, technological advancement has dramatically increased the production of nanometer-sized particles or nanoparticles (NPs), and many epidemiological studies have confirmed a correlation between NP exposure and the onset of cardiovascular diseases and various cancers. Among the adverse effects on human health, in recent years, potential hazards of nanomaterials on female reproductive organs have received increasing concern. Several animal and human studies have shown that NPs can translocate to the ovary, uterus, and placenta, thus negatively impacting female reproductive potential and fetal health. However, NPs are increasingly being used for therapeutic purposes as tools capable of modifying the natural history of degenerative diseases. Here we briefly summarize the toxic effects of few but widely diffused NPs on female fertility and also the use of nanotechnologies as a new molecular approach for either specific pathological conditions, such as ovarian cancer and infertility, or the cryopreservation of gametes and embryos.

Controlled ovarian stimulation (COS) through gonadotropin administration has become a common procedure in assisted reproductive technologies. COS’s drawback is the formation of an unbalanced hormonal and molecular environment that could alter several cellular mechanisms. On this basis, we detected the presence of mitochondrial DNA (mtDNA) fragmentation, antioxidant enzymes (catalase; superoxide dismutases 1 and 2, SOD-1 and -2; glutathione peroxidase 1, GPx1) and apoptotic (Bcl-2-associated X protein, Bax; cleaved caspases 3 and 7; phosphorylated (p)-heat shock protein 27, p-HSP27) and cell-cycle-related proteins (p-p38 mitogen-activated protein kinase, p-p38 MAPK; p-MAPK activated protein kinase 2, p-MAPKAPK2; p-stress-activated protein kinase/Jun amino-terminal kinase, p-SAPK/JNK; p-c-Jun) in the oviducts of unstimulated (Ctr) and repeatedly hyperstimulated (eight rounds, 8R) mice. While all the antioxidant enzymes were overexpressed after 8R of stimulation, mtDNA fragmentation decreased in the 8R group, denoting a present yet controlled imbalance in the antioxidant machinery. Apoptotic proteins were not overexpressed, except for a sharp increase in the inflammatory-related cleaved caspase 7, accompanied by a significant decrease in p-HSP27 content. On the other hand, the number of proteins involved in pro-survival mechanisms, such as p-p38 MAPK, p-SAPK/JNK and p-c-Jun, increased almost 50% in the 8R group. Altogether, the present results demonstrate that repeated stimulations cause the activation of the antioxidant machinery in mouse oviducts; however, this is not sufficient to induce apoptosis, and is efficiently counterbalanced by activation of pro-survival proteins.

Molecular biology techniques are increasingly being used in sex identification of skeletal remains when traditional anthropometric analyzes are not successful in identifying sex of remains that are incomplete, fragmented and /or of immature individuals. In the present work, we investigated the possibility of determining sex by using the qPCR-duplex method for both ancient and modern DNA samples. This method involves the co-amplification of two genes in a single reaction system and the subsequent analysis of the fusion curves; the gene sequences used for the construction of suitable primers are those of steroid sulfatase (STS) and testis specific protein Y-linked 1 (TSPY) genes which turned out to be two sensitive markers as they have a detection limit of 60 pg and 20 pg respectively on modern DNA. The validity of the method was verified on modern DNA in which gender was identified in all the samples with 100% accuracy; thus, allowing for the same results as the classic method with amelogenin, but in a faster and more immediate way, as it allows for sex determination solely by analyzing the denaturation curves without having to perform an electrophoretic run. The proposed molecular technique proves to be sensitive and precise even on degraded DNA, in fact on 9 archaeological finds dating from the VII-XII century in which sex had been identified through anthropometric analysis, it confirmed the sex of 8 out of 9 finds correctly.

The potential risks of environmental nanoparticles (NPs), in particular Polystyrene Nanoparticles (PNPs), is an emerging problem; specifically, the interaction of PNPs with intestinal cells has not been characterized so far. The mechanism by which polystyrene particles are transferred to humans has not yet been clarified, whether directly through ingestion from contaminated food. We evaluated the interaction between PNPs and colorectal adenocarcinoma cells (HCT116). Cells were exposed to different concentrations of PNPs, metabolic activity and the consequent cytotoxic potential were assessed through viability test; we evaluated the PNP genotoxic potential through the Cytokinesis-Block Micronucleus cytome (CBMN cyt) assay. Finally, we detected Reactive Oxygen Species (ROS) production after NPs exposure and performed Western Blot analysis to analyze the enzymes (SOD1, SOD2, Catalase, Glutathione Peroxidase) involved in the cell detoxification process that comes into play during the cell-PNPs interaction. This work analyzes the cyto and genotoxicity of PNPs in the colorectal HCT116 cell line, in particular the potential damage from oxidative stress produced by PNPs inside the cells related to the consequent nuclear damage. Our results show moderate toxicity of PNPs both in terms of ROS production and DNA damage. Further studies will be needed on different cell lines to have a more complete picture of the impact of environmental pollution on human health in terms of PNPs cytotoxicity and genotoxicity.

Aim The city of L'Aquila (central Italy) was hit by a strong earthquake in 2009 that caused the collapse of several buildings, deaths and injured people. In the following years, a great number of building sites were activated, building workers resulted intensely exposed and represent a relevant target for research on environmental mutagenesis and epidemiological surveillance. Cells of buccal mucosa are considered an appropriate site for early detecting of cytogenetic damage, since it represents the first barrier in inhalation or ingestion and can metabolize carcinogenic agents into reactive chemicals. Our study is aimed 1) at comparing the early genotoxic damage as measured by the buccal mucosa micronucleus test in two subgroups of workers defined by different occupational exposure and 2) at evaluating possible confounding variables such as lifestyle factors. Methods and results A cross-sectional study was conducted in L'Aquila, on 24 outdoor workers (OWs) highly exposed on the construction sites and 26 indoor workers (IWs), all subjected to the compulsory occupational surveillance system, in the period 2017–2018. Buccal cells samples were collected and, based on the Micronucleus test, the exfoliated cells were classified in respect of nuclear changes observed. Moreover, a self-report questionnaire composed of 84 items, was administered to the workers. Results Significant differences were observed between Exp+ (OWs) and Exp− (IWs) in the number of the analyzed cells (expressed as mean value out of 1000 cells): respectively 954.46 vs 990.06 normal cells, (p Conclusion Despite some limitation, our findings clearly confirm the role of occupational exposure as a marker of cytogenetic damage associated with MNs number in construction workers. Moreover, smoking status appears as the only other investigated factor independently associated to the outcome. The statistical model, in addition, highlights possible moderation and confounding effects, such as interaction between smoking and occupational exposure and the unbalanced school education level in workers. Micronucleus test in exfoliated buccal cells would be considered a suitable method for studying the early genotoxic damage in the construction occupational setting as well as in evaluating the efficacy of preventive practices.

Study of ancient DNA makes it possible to analyze genetic relationships between individuals and populations of past and present. In this paper we have analyzed remains of human bones, dating back to the 8th-10th century AD, from the burials found in the Cathedral of Santa Maria in Civitate, archaeological site of Amiternum, L'Aquila, Italy. As a genetic marker, the hypervariable region 1 of mitochondrial DNA (HVR1) was selected. To obtain reliable sequences from the hypervariable region 1 of mtDNA (HVR1) were performed: multiple extractions, template quantification and cloning of PCR products. The sequences obtained were compared with Anderson's sequence for the identification of polymorphisms (SNP) and haplogroups. The data obtained were analyzed with various software and phylogenetic methods. For the comparison between populations, ancient and modern sequences found in databases and literature have been used. This work provides preliminary information on the correlation between the population of Amiternum, the migrant populations transited and/or established in the territory of Amiternum such as Byzantines, Longobards (Lombards), which dominated the Italian peninsula between 568 and 774 AD, and the current populations of Italy. The study of haplogroups, the analysis of genetic variability and phylogenesis studies on the sequences considered show a genetic closeness between the individuals of Amiternum, the current population of central-northern Italy and the Germanic tribe of Longobards, however, also highlights genetic traits of Byzantines in some samples of Amiternum. Using the analysis of amelogenin gene fragments, we successfully determined the sex of the bone remains on all samples., Genetics; DNA sequencing; Mutation; Phylogeny; Archeology; Ancient DNA; Amiternum; Lombard; Amelogenin gene; mtDNA

A number of studies have shown variable grades of cytotoxicity and genotoxicity in in vitro cell cultures, laboratory animals and humans when directly exposed to particle debris generated from tires. However, no study has compared the effects of particles generated from passenger tires with the effects of particles from truck tires. The aim of this study was to investigate and relate the cyto- and genotoxic effects of different types of particles (PP, passenger tire particles vs. TP, truck tire particles) in vitro using the phagocytic cell line RAW 264.7 (mouse leukaemic monocyte macrophage cell line). The viability of RAW 264.7 cells was determined by the 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS) assay following exposure for 4, 24 and 48 hours to different particle concentrations (10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml). The effects of particles of passenger and truck tires on cell proliferation and genotoxicity were evaluated by means of the cytokinesis-block micronucleus (CBMN) assay following exposure for 24 hours to different particle concentrations (10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml). In MTS assay, after 24 hours, it was found that PP induced a 30% decrease in metabolic activity at a concentration of 10 μg/ml, while TP caused reductions of 20% and 10% at concentrations of 10 μg/ml and 50 μg/ml, respectively. At 48 hours after the treatments, we observed increased metabolic activity at 50 μg/ml and 100 μg/ml for the PP while only at 50 μg/ml for the TP. The CBMN assay showed a significant increase in the number of micronuclei in the cells incubated with PP in all experimental conditions, while the cells treated with TP showed a meaningful increase only at 10 μg /ml. We utilized the TNF-α ELISA mouse test to detect the production of tumour necrosis factor-alpha (TNF-α) in RAW 264.7 cells. The effect of passenger and truck particles on TNF-α release was evaluated following exposure for 4 and 24 hours. After 4 hours of incubation, the cells treated with PP and TP at 100 μg / ml showed a slight but significant increase in TNF-α release, while there was a significant increase in the release of TNF-α after 24 hours of incubation with both tire samples in the cells treated with 50 and 100 μg / ml PP. The data obtained show a higher cytotoxic, clastogenic/genotoxic and inflammatory effects of passenger compared to the truck tire particles.

Several studies have provided information on environmental nanoplastic particles/debris, but the in vitro cyto-genotoxicity is still insufficiently characterized. The aim of this study is to analyze the effects of polystyrene nanoparticles (PNPs) in the Hs27 cell line. The viability of Hs27 cells was determined following exposure at different time windows and PNP concentrations. The genotoxic effects of the PNPs were evaluated by the cytokinesis-block micronucleus (CBMN) assay after exposure to PNPs. We performed ROS analysis on HS27 cells to detect reactive oxygen species at different times and treatments in the presence of PNPs alone and PNPs added to the Crocus sativus L. extract. The different parameters of the CBMN test showed DNA damage, resulting in the increased formation of micronuclei and nuclear buds. We noted a greater increase in ROS production in the short treatment times, in contrast, PNPs added to Crocus sativus showed the ability to extract, thus reducing ROS production. Finally, the SEM-EDX analysis showed a three-dimensional structure of the PNPs with an elemental composition given by C and O. This work defines PNP toxicity resulting in DNA damage and underlines the emerging problem of polystyrene nanoparticles, which extends transversely from the environment to humans, further studies are needed to clarify the internalization process.

A survey of the truffle Tuber melanosporum genome has shown the presence of 67 programmed cell death (PCD)-related genes. The 67 genes are all expressed during fruit body (FB) development of T. melanosporum development; their expression has been detected by DNA microarrays and qPCR. A set of 14 PCD-related genes have been chosen, those with the highest identities to the homologs of other species, for a deeper investigation. That PCD occurs during T. melanosporum development has been demonstrated by the TUNEL reaction and transmission electron microscopy. The findings of this work, in addition to the discovery of PCD-related genes in the T. melanosporum genome and their expression during the differentiation and development of the FB, would suggest that one of the PCD subroutines, maybe autophagy, is involved in the FB ripening, i.e., sporogenesis.

Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn't differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication.

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17–18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O2 consumption during B. bufo development. Keywords: Amphibian egg, Tyrosinase, Pigment cell, Melanin, Oxygen

Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors.

The symbiotic fungus Tuber melanosporum Vittad. (Perigord black truffle) belongs to the Ascomycota and forms mutualistic symbiosis with tree and shrub roots. This truffle has a high value in a global market and is cultivated in many countries of both hemispheres. The publication of the T. melanosporum genome has given researchers unique opportunities to learn more about the biology of the fungus. Real-time quantitative PCR (qRT-PCR) is a definitive technique for quantitating differences in transcriptional gene expression levels between samples. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes must show stable expression under given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in T. melanosporum development. In this study, we present a morphological and microscopical classification of the developmental stages of T. melanosporum fruit body, and investigate the expression levels of 12 candidate reference genes (18S rRNA; 5.8S rRNA; Elongation factor 1-alpha; Elongation factor 1-beta; α-tubulin; 60S ribosomal protein L29; β-tubulin; 40S ribosomal protein S1; 40S ribosomal protein S3; Glucose-6-phosphate dehydrogenase; β-actin; Ubiquitin-conjugating enzyme). To evaluate the suitability of these genes as endogenous controls, five software-based approaches and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. We demonstrate here that the 18S rRNA gene shows the most stable expression during T. melanosporum development and that a set of three genes, 18S rRNA, Elongation factor 1-alpha and 40S ribosomal protein S3, is the most suitable to normalize qRT-PCR data from all the analyzed developmental stages; conversely, 18S rRNA, Glucose-6-phosphate dehydrogenase and Elongation factor 1-alpha are the most suitable genes for fruiting body developmental stages.

Methylglyoxal is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Recent research indicates that methylglyoxal is a potent growth inhibitor and genotoxic agent. The antiproliferative activity of methylglyoxal has been investigated for pharmacological application in cancer chemotherapy. However, various cells are not equally sensitive to methylglyoxal toxicity. Therefore, it would be important to establish the cellular factors responsible for the different cell-type specific response to methylglyoxal injury, in order to avoid the risk of failure of a therapy based on increasing the intracellular level of methylglyoxal. To this purpose, we comparatively evaluated the signaling transduction pathway elicited by methylglyoxal in human glioblastoma (ADF) and neuroblastoma (SH-SY 5Y) cells. Results show that methylglyoxal causes early and extensive reactive oxygen species generation in both cell lines. However, SH-SY 5Y cells show higher sensitivity to methylglyoxal challenge due to a defective antioxidant and detoxifying ability that, preventing these cells from an efficient scavenging action, elicits extensive caspase-9 dependent apoptosis. These data emphasize the pivotal role of antioxidant and detoxifying systems in determining the grade of sensitivity of cells to methylglyoxal.

Nanomaterials are present in a number of commercially available products but there are uncertainties as to whether the unique properties that support their commercial use may also pose potential health risks. Information is missing concerning the influence of nanomaterials on the overall reproductive outcome and transgenerational effects in animals and plants. To obtain this information, long-term studies would be required using animal models phylogenetically close to humans and exposure conditions that reflect realistic scenarios with regard to dosages and admission. The nanoreprotoxicology literature published to date is largely descriptive in nature regarding the effects of nanoparticles. The mechanisms, which determine particle reproduction compatibility, are mostly elusive at the moment. Thus, it is recommended that future research explore the interactions between nanomaterials and transgenerational matter on a molecular level. It would, for instance, be of major importance to understand the behaviour of nanoparticles inside the cells but also their genotoxic and epigenetic effects. Recent studies have shown that intravenous and/or intra-abdominal administration of nanoparticles to mice results in their accumulation in the cells of many tissues, including the brain and the testis, suggesting that they easily pass through the blood-brain and blood-testis barriers. In parallel embryo development after exposure to nanoparticles should be comparatively investigated. The majority of studies on embryo toxicology have concentrated on piscine embryos, mostly derived from zebrafish. Plants for human food as an important component of the ecosystem need also to be taken into account when evaluating transgenerational effects of engineered nanomaterials in crops.

Nanomaterials are present in a number of commercially available products but there are uncertainties as to whether the unique properties that support their commercial use may also pose potential health risks. Information is missing concerning the influence of nanomaterials on the overall reproductive outcome and transgenerational effects in animals and plants. To obtain this information, long-term studies would be required using animal models phylogenetically close to humans and exposure conditions that reflect realistic scenarios with regard to dosages and admission. The nanoreprotoxicology literature published to date is largely descriptive in nature regarding the effects of nanoparticles. The mechanisms, which determine particle reproduction compatibility, are mostly elusive at the moment. Thus, it is recommended that future research explore the interactions between nanomaterials and transgenerational matter on a molecular level. It would, for instance, be of major importance to understand the behaviour of nanoparticles inside the cells but also their genotoxic and epigenetic effects. Recent studies have shown that intravenous and/or intra-abdominal administration of nanoparticles to mice results in their accumulation in the cells of many tissues, including the brain and the testis, suggesting that they easily pass through the blood–brain and blood–testis barriers. In parallel embryo development after exposure to nanoparticles should be comparatively investigated. The majority of studies on embryo toxicology have concentrated on piscine embryos, mostly derived from zebrafish. Plants for human food as an important component of the ecosystem need also to be taken into account when evaluating transgenerational effects of engineered nanomaterials in crops.

Background It has been proposed that the anticonvulsant drug phenytoin (PHT) requires bioactivation to reactive intermediate(s) to achieve its recognized teratogenic potential and that embryonal detoxification power may play a fundamental role in the teratogenic response. On this basis, we sought to investigate the potential effects of a teratogenic exposure to PHT on the activities of antioxidant and GSH-related detoxifying enzymes in gestational murine tissues. Methods Pregnant Swiss mice were injected intraperitoneally with 0 (vehicle) or 65 mg/kg of PHT on gestation day (GD) 12 (plug day = GD 1). Biochemical determinations, including activities of glutathione transferase, glutathione peroxidase, glutathione reductase, glyoxalase I, glyoxalase II, catalase, and superoxide dismutase, were carried out on maternal and embryonic/fetal livers and in placentas on GD 14 and 19. Results The major findings of this study show that (1) organogenesis-stage conceptal tissues have detectable levels of all the tested enzymes; (2) most of the embryonic liver and placental enzymes investigated undergo a significant induction within 48 hr (GD 14) after PHT administration; and (3) in the same tissues a down-regulation of enzyme activities is noted near term (GD 19). Conclusions Overall, these findings show that teratogenic exposure to PHT is associated with a modulation of reactive-intermediates-scavenging enzyme activities, and provide further support for role of generation of reactive intermediates in PHT-induced teratogenesis. Teratology 62:100–107, 2000. © 2000 Wiley-Liss, Inc.

The cDNAs of Tuber melanosporum laccases (Tmellcc1 and Tmellcc2) have been cloned. From the cloned cDNAs probes were prepared to investigate the expression levels of the Tmellcc1 and Tmellcc2 genes in the free living mycelium (FLM), ectomycorrhizae (ECM) and different developmental stages of fruit body (FB) by quantitative PCR (qPCR). The mRNA expression levels agree with the changes of laccase activities. The histochemical data agree with the qPCR and biochemical results. The highest laccase expression occurs in the ECM, when the host plant roots are invaded by the fungal mycelium.

The present work studies the expression of tyrosinase (monophenol:diphenol oxygen oxidoreductase, EC 1.14.18.1) during the development of the black truffle Tuber melanosporum Vittad., an ectomycorrhizal fungus of great biological and economic interest. As widely reported in the literature, melanins and the enzymes that synthesize them, are of paramount importance in fungal development and sexual differentiation. Tyrosinase and laccase are the enzymes that produce melanins from monophenols and diphenols. We have detected tyrosinase expression from the stage of free living mycelium, through the mychorrizal stage and the six fruit body developmental stages by measuring the levels of tyrosinase mRNA by quantitative PCR (q-PCR), spectrophotometry, histochemistry, immunohistochemistry and electrophoresis. Tyrosinase is always expressed, from the free living mycelium to the ripe fruit body developmental stages, when it is very low. The switching off of the tyrosinase gene during T. melanosporum development when the fruit body is ripe and no more cell walls are to be built is discussed in relation of thioflavour production. Specific primers, prepared from the cloned T. melanosporum tyrosinase cDNA were used for the q-PCR and the deduced aminoacid sequences of the CuA and CuB binding sites were compared to those of various ascomycetes and basidiomycetes.

Recent data from literature report that reactive oxygen species (ROS) seem to play a crucial role in the etiology of both types I and II diabetes. This may render diabetic individuals more prone to oxidative injury when challenged with hypoxic stress. It is in fact well known that many diabetic complications cause ischaemic episodes, with a consequent reduction in oxygen supply to various tissues and organs. To check this hypothesis, in this work we tested type I diabetic individuals’ antioxidant capability towards a hypoxic-mediated oxidative challenge. In particular, spontaneously diabetic and age-matched non-diabetic biobreeding (BB) Wistar rats were submitted to chronic normobaric hypoxia, and the response of antioxidant enzymes, as well as redox-sensitive transcription factor NF-κB and p53, were monitored. Results show that diabetic subjects present a dramatic enhancement in the major antioxidant enzymes activities, thus supporting the notion of diabetes-related changes in cellular redox status. This allows diabetic individuals to counteract hypoxia-mediated oxidative challenge better than the non-diabetic counterpart. Also the behaviour of both the redox-sensitive nuclear transcription factor NF-κB and p53 protein in response to hypoxic stimulation seems to support the hypothesis of a better ROS scavenging efficiency in diabetics under hypoxic conditions. In conclusion, high levels of antioxidant enzymatic defences in diabetic BB rats reflect a positive adaptive response able to assure an efficient protection not only against chronic, diabetes-mediated reactive oxygen species (ROS) overproduction, but also versus further oxidative damage.

Melanocytes and neuroblasts share the property of transforming L-tyrosine through two distinct metabolic pathways leading to melanogenesis and catecholamine synthesis, respectively. While tyrosinase (TYR) activity has been shown to be expressed by neuroblastoma it remains to be established as to whether also glioblastomas cells are endowed with this property. We have addressed this issue using the human continuous glioblastoma cell line ADF. We demonstrated that these cells possess tyrosinase as well as L-tyrosine hydroxylase (TH) activity and synthesize melanosomes. Because the two pathways are potentially cyto-genotoxic due to production of quinones, semiquinones, and reactive oxygen species (ROS), we have also investigated the expression of the peroxisomal proliferators activated receptor alpha (PPARalpha) and nuclear factor-kB (NFkB) transcription factor as well the effect of L-tyrosine concentration on cell survival. We report that L-tyrosine down-regulates PPARalpha expression in ADF cells but not neuroblastoma and that this aminoacid and phenylthiourea (PTU) induces apoptosis in glioblastoma and neuroblastoma.

Conjugated linoleic acid (CLA) has been shown to exert beneficial effects against carcinogenesis, atherosclerosis and diabetes. It has been demonstrated that CLA modulates lipid metabolism through the activation of peroxisome proliferator-activated receptors (PPARs). The PPAR family comprises 3 closely related gene products, PPAR alpha, beta/delta and gamma, differing for tissue distribution, developmental expression and ligand specificity. It has also been demonstrated that activated PPARgamma results in growth inhibition and differentiation of transformed cells. These observations stimulated a great interest toward PPARgamma ligands as potential anticancer drugs to be used in a differentiation therapy. Glioblastomas are the most commonly diagnosed primary tumors of the brain in humans. The prognosis of patients with high-grade gliomas is poor and only marginally improved by chemotherapy. The aim of this work was to study the effects of CLA and of a specific synthetic PPARgamma ligand on cell growth, differentiation and death of a human glioblastoma cell line as well as on parameters responsible for the metastatic behavior of this tumor. We demonstrate here that CLA and PPARgamma agonist strongly inhibit cell growth and proliferation rate and induce apoptosis. Moreover, both treatments decrease cell migration and invasiveness. The results obtained show that CLA acts, directly or indirectly, as a PPARgamma activator, strongly suggesting that this naturally occurring fatty acid may be used as brain antitumor drug and as a chemopreventive agent. Moreover, the gamma-agonist, once experimented and validated on man, may represent a useful coadjuvant in glioblastoma therapy and in the prevention of recurrences.

The filametous ascomycete Tuber borchii is a plant-symbiotic ectomycorrhizal microrganism with an high value due to the production of hypogeous fruitbodies (truffles). The present work was undertaken to develop a procedure for the release of T. borchii viable protoplasts from Tuber mycelium, isolate ATTC 96540; several factors which affect the isolation, morphology and viability were examined and developed in order to improve applications of T. borchii protoplasts in morphological, biochemical and genetic investigations (protoplast fusion or transformation). Functional delivery of liposome content into T. borchii protoplasts has also been examined with a cytotoxic ribosome inactivator as saporin. T. borchii protoplasts incubation/fusion with saporin containing liposomes were made to demonstrate the absence of cell wall of 16 days cultured protoplasts.

Methylglyoxal (MG) is one of the most powerful glycating agents of proteins and other important cellular components and has been shown to be toxic to cultured cells. Under hyperglycaemic conditions, an increase in the concentration of MG has been observed in human body fluids and tissues that seems to be responsible for diabetic complications. Recent data suggest that diabetes may cause impairment of cognitive processes, according to a mechanism involving both oxidative stress and advanced glycation end product (AGE) formation. In this work, we explored the molecular mechanism underlying MG toxicity in neural cells, by investigating the effect of MG on both the interleukin-1beta (IL-1beta), as the major inducer of the acute phase response, and the nervous growth factor (NGF) expression. Experiments were performed on cultured neural cells from rat hippocampus, being this brain region mostly involved in cognitive processes and, therefore, possible target of diabetes-mediated impairment of cognitive abilities. Results show that MG treatment causes in hippocampal neural cells extensive, oxidative stress-mediated cell death, in consequence of a strong catalase enzymatic activity and protein inhibition. MG also causes a very significant increase in both transcript and protein expression of the NGF as well as of the pro-inflammatory cytokine IL-1beta. MG co-treatment with the antioxidant N-acetylcysteine (NAC) completely abrogates the observed effects. Taken together, these data demonstrate that hippocampal neurons are strongly susceptible to MG-mediated oxidative stress.

We present the results of an experiment aimed at comparing the effects of different background radiation environments on metabolism and responses to gamma-rays and cycloheximide of cultured mammalian cells. Chinese hamster V79 cells were maintained in exponential growth in parallel for up to 9 months at the Istituto Superiore di Sanita (ISS) and at the INFN-Gran Sasso underground Laboratory (LNGS) where exposure due to gamma-rays and to radon was reduced by factors of about 70 and 25, respectively. After 9 months the cells grown at the LNGS (cumulative gamma dose about 30 microGy, average radon concentration around 5 Bq/m(3)), compared to the cells grown at the ISS (cumulative gamma-ray dose about 2 mGy, average radon concentration around 120 Bq/m(3)), exhibited i). a significant increase of the cell density at confluence, ii). a significantly higher capacity to scavenge organic and inorganic hydroperoxides but a reduced scavenging capacity towards superoxide anions and iii). an increase in both the basal hprt mutation frequency and sensitivity to the mutagenic effect of gamma-rays. The cells grown at the LNGS also showed a greater apoptotic sensitivity starting at the third month of culture, that was no longer detected after 9 months. Overall, these data suggest a role of background ionizing radiation in determining an adaptive response, although they cannot be considered conclusive.

Methylglyoxal (2-oxopropanal) is a reactive alpha-oxoaldehyde that can be formed endogenously mainly as a by-product of glycolytic pathway. It is a cytotoxic compound with significant antiproliferative properties as it can bind, under physiological conditions, to nucleic acids and proteins, forming stable adducts. We have recently shown that exogenous methylglyoxal (150-600 microM) is highly toxic for amphibian embryos where it produces, when added to the culture water, inhibition of cell proliferation in the early developmental stages, followed by severe malformations and strongly reduced embryonic viability. In this work we investigate the morphofunctional effect of methylglyoxal on the common toad B. bufo embryo mitochondria in order to verify if its dysmorphogenetic action might be also ascribed to impairment of mitochondrial functions. The mitochondria were isolated from embryos at the developmental stages of morula, neural plate and operculum complete and developing in the presence of 600 microM methylglyoxal. The results show that exogenous methylglyoxal is highly toxic at mitochondrial level, where it produces proliferation, swelling and membrane derangement. As a consequence, mitochondria from treated embryos show decreased oxidative phosphorylation efficiency, as indicated by the significant reduction both of the respiratory control index values and of the embryonic ATP content. On the basis of these data, it is possible that the methylglyoxal-induced embryonic malformations as well as the strongly reduced viability might be also ascribed to energy depletion.

This work deals with the antioxidant enzymatic response and the ultrastructural aspects of the skeletal muscle of young and aged rats kept under hypoxic or hyperoxic normobaric conditions. It is in fact well known that the supply of oxygen at concentrations higher or lower than those occurring under normal conditions can promote oxidative processes that can cause tissue damage. The enzymes investigated were both those directly involved in reactive oxygen species (ROS) scavenging (superoxide dismutase, catalase and selenium-dependent glutathione peroxidase), and those challenged with the detoxication of cytotoxic compounds produced by the action of ROS on biological molecules (glutathione transferase, glyoxalase I, glutathione reductase), in order to obtain a comparative view of the defence strategies used with respect to aging. Our results support the hypothesis that one of the major contributors to the aging process is the oxidative damage produced at least in part by an impairment of the antioxidant enzymatic system. This makes the aged organism particularly susceptible to oxidative stress injury and to the related degenerative diseases, especially in those tissues with high demand for oxidative metabolism.

Truffles live in an environment poor in oxygen, and are a good model for investigation of enzymatic adaptation to microaerobic conditions. We studied the antioxidant and glutathione dependent enzymatic endowment of Tuber truffles. Superoxide dismutase, catalase, glutathione peroxidase Se dependent, glutathione reductase, glyoxalase 1 and glyoxalase 2 are expressed and correlate with the microaerobic metabolism, growth rate and mycorrhizal symbiosis of truffles. A very low or undetectable glutathione S-transferase activity was found. For comparison the same enzyme activities were investigated in Agaricus bisporus which is epigeous and non-mycorrhizal.

This paper reports the effect of the tyrosinase (monophenol o-diphenol:oxygen oxidoreductase; EC 1.14.18.1) inhibitors diethyldithiocarbamate (DETC), L-tropolone, kojic acid, phenylthiourea (PTU) and L-mimosine on the in vitro growth of Tuber borchii (a white truffle) mycelium. A significant inhibitory effect on mycelium growth was observed for DETC, PTU and L-tropolone (0% growth compared to control at 100 microg ml(-1) DETC, PTU and L-tropolone and at 10 microg ml(-1) DETC and L-tropolone). As a comparison the action of the same inhibitors was also tested on the growth and pigmentation of the mould Cladosporium sphaerospermum. In the presence of CuSO(4) 10(-6) M T. borchii mycelium acquired pigmentation (as rounded aggregates compared to control revealed by SEM microscopy). Tyrosinase activity in the extract from T. borchii mycelium (18-day culture) was detected spectrophotometrically.

Methylglyoxal (2-oxopropanal) is a cytotoxic compound that can be formed endogenously as a by-product of glycolytic pathway; so its concentration is expected to increase when glycolysis activity increases such as during embryo development. In this work we study the effect of exogenous methylglyoxal on development and embryo viability during Bufo Bufo development and on the enzymes and cofactors involved in its detoxication process (glyoxalase I and II, reduced glutathione and glyceraldehyde 3-phosphate dehydrogenase). The results show that exogenous methylglyoxal does not affect the enzymatic pattern until stage 20, while it induces a significant activity decrease of the tested enzymes at stage 25. On the contrary methylglyoxal positively influences the reduced glutathione concentration at all the considered stages. At morphological and histological levels methylglyoxal causes a strong retardation of cell division in the early stages, that results in various abnormalities in the late development. In conclusion, methylglyoxal enters the embryo and is antiproliferative and teratogenic: the data further supports the hypothesis of the importance of the glyoxalase system in the process of cell growth and division.

In this work we have investigated the expression of glyoxalase I (GLO I) and glyoxalase II (GLO II) activities during Bufo bufo embryo development and in some tissues of both male and female adult animals, in order to study how they correlate with cell proliferation and differentiation. The results show that both the activities are expressed at significant levels from the earliest developmental stages, reaching the highest values at the end of embryonic development (stage 25). The GLO I/GLO II ratio is very high at the beginning of the development and then gradually decreases as the development goes on. These data emphasize the importance of GLO I activity in the phases in which elevated cell division is taking place. In the differentiated tissues, a peculiar sexual dimorphism in both GLO I and GLO II activities, with higher values in female than in male, was found. GLO I embryonic activity levels are comparable to those found in female differentiated tissues, but significantly higher than those detected in male differentiated tissues. On the contrary, the GLO II activities found in the adult tissues were always higher than those found in embryos. These results further support the idea that high GLO I/GLO II ratios are a characteristic of the proliferative status, which assures a good scavenging action against the potentially cytotoxic and cytostatic effect of methylglyoxal.

Mammalian cells were cultured in parallel at the Istituto Superiore di Sanita', in the presence of standard radiation background, and at the INFN-Gran Sasso underground Laboratory, exposed to low radiation background. Both cultures were grown for up to six months and periodically tested to check for the onset of metabolic changes and different sensitivity to genotoxic damage caused by ionizing and UV radiation, as well as by chemical agents. These tests include measurements of enzyme activities protecting from oxidative cell damage, induction of apoptosis and mutation.